Method for muscle delivery of drugs, nucleic acids and other compounds

ABSTRACT

A method is disclosed for delivering molecules such as pharmaceutical drugs and nucleic acids into skeletal muscle in vivo. The molecule is first injected into the muscle at one or multiple sites. Immediately or shortly after injection, electrodes are placed flanking the injection site and a specific amount of electrical current is passed through the muscle. The electrical current makes the muscle permeable, thus allowing the molecule to enter the cell. In the case where nucleic acid is injected, the efficiency of transfer permits expression of protein encoded by the nucleic acid in an amount that exhibits systemic biological activity and which generates a robust immune response.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

[0001] This application is a continuation of copending U.S. patentapplication Ser. No. 10/141,561 filed May 7, 2002, and entitled “Methodfor Genetic Immunization” which is a continuation of copending U.S.patent application Ser. No. 09/899,561 filed Jul. 5, 2001, and entitled“Method for Genetic Immunization and Introduction of Molecules intoSkeletal Muscle and Immune Cells,” which is a continuation of U.S.application Ser. No. 09/565,140 (now U.S. Pat. No. 6,261,281), filed May5, 2000 and entitled “Method for Genetic Immunization and Introductionof Molecules into Skeletal Muscle and Immune Cells”, which is acontinuation-in-part of U.S. patent application Ser. No. 09/055,084 (nowU.S. Pat. No. 6,110,161), filed Apr. 3, 1998, and entitled “Method forIntroducing Pharmaceutical Drugs and Nucleic Acids Into SkeletalMuscle,” which is related to and claims the benefit of U.S. ProvisionalApplication Serial No. 60/042,594, filed Apr. 3, 1997, and entitled“Apparatus and Method for Introducing Pharmaceutical Drugs and GeneticMaterial Into Skeletal Muscle.” These applications are herebyincorporated by reference.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention is related to a method for making skeletalmuscle semipermeable to pharmaceutical drugs, nucleic acids and othermolecules. More specifically, skeletal muscle is made semipermeable byelectrically stimulating the muscle with a current that generates lowfield strengths following injection of pharmaceutical drugs, nucleicacids and other molecules. The method can be used to obtain expressionof proteins encoded by nucleic acids administered to muscle inaccordance with the method.

[0004] 2. Technical Background

[0005] Scientists are continually discovering genes which areresponsible for many human diseases, such as genes responsible for someforms of breast cancer, colon cancer, muscular dystrophy and cysticfibrosis, In addition, scientists are continually discovering genes thatcode for bacterial and viral antigens (e.g., viral capsid proteins).Despite these new discoveries, a major obstacle facing the medicalprofession is how to safely deliver effective quantities of these agentsto patients to treat disease or for genetic immunization.

[0006] Currently, most pharmaceutical agents are taken orally orintravenously. Oral and intravenous drug and gene delivery methods,however, have several shortcomings. First, a large percent of orally orintravenously delivered drugs are degraded by the body before arrivingat the target organ or cells. Acids and enzymes in the stomach andintestine, for example, can break down many pharmaceutical drugs.Similarly, genes would be rapidly destroyed by proteins found in theblood and liver which break down DNA. Additionally, intravenouslydelivered drugs and genes are often sequestered by the liver or immunesystem before arriving at the diseased organ or cells. Second, oral andintravenous drug and gene delivery is non-specific. That is, the drug orgene is delivered to both target and non-target cells.

[0007] Skeletal muscle is a promising candidate for drug delivery, genetherapy and genetic immunization. First, skeletal muscle constitutesover 50% of a human's body mass, most of which is easily accessiblecompared to other tissues and organs of the body. Second, there arenumerous inherited and acquired disorders, such as Duchenne musculardystrophy (DMD), diabetes mellitus, hyperlipidaemia and cardiovasculardisease which are good candidate disorders for drug and gene deliveryinto the muscle. Third, muscle is an ideal site for genetic immunizationbecause it is easily accessible and proteins made in the muscle aresecreted, thus eliciting an immune response. Finally, skeletal musclecells are non-dividing. Therefore, skeletal muscle cells are capable ofexpressing a protein coded by a gene for a longer time period than wouldbe expected of other cell types that are continually dividing. Becausethe protein is expressed for a longer time, fewer treatments would benecessary.

[0008] Currently, however, there is no non-viral method for effectivelydelivering pharmaceutical drugs, proteins, and DNA into skeletal musclein vivo. There are several methods known in the art for transferringpharmaceutical drugs and DNA into skeletal muscle, such as intramuscularinjection of DNA. The clinical applicability of direct muscle injection,however, is limited mainly because of low transfection efficiency,typically less than 1% transfection efficiency. It has been demonstratedthat the efficacy of transfection can be improved if DNA injections aredone in regenerating muscle. Regeneration is induced three days beforeDNA injection with the drug Bivucain. While injection in regeneratingmuscles induced by Bivucain show higher efficiency, the method haslimited applicability in humans because of the severe damage caused tothe muscle.

[0009] From the foregoing, it will be appreciated that it would be anadvancement in the art to provide a non-viral method of deliveringpharmaceutical drugs and DNA only to diseased organs and cells. It wouldalso be an advancement in the art to provide an electroporation methodof delivering pharmaceutical drugs and DNA directly into skeletalmuscle. It would be yet another advancement in the art if theelectroporation method could deliver therapeutically effectivequantities of pharmaceutical drugs and DNA into the skeletal muscle atmultiple sites simultaneously. It would be a further advancement if themethod permitted the delivery efficiencies to be regulated.

[0010] Such a method is disclosed herein.

SUMMARY OF THE INVENTION

[0011] The present invention provides a method for delivering ortransfecting pharmaceutical drugs, proteins, and DNA into skeletalmuscle and other cells residing within the skeletal muscle. Withoutbeing bound by theory, the method is thought to be similar toelectroporation. Electroporation works on the principle that a cell actsas an electrical capacitor and is generally unable to pass current.Subjecting cells to a high-voltage electric field, therefore, createstransient permeable structures or micropores in the cell membrane. Thesepores are large enough to allow pharmaceutical drugs, DNA, and otherpolar compounds to gain access to the interior of the cell. With time,the pores in the cell membrane close and the cell once again becomesimpermeable.

[0012] Conventional electroporation, however, employs high fieldstrengths from 0.4 to several kV/cm. In contrast to conventionalelectroporation, the field strength used in the present invention rangesfrom about 10 V/cm to about 300 V/cm. These lower field strengths arethought to cause less muscle damage without sacrificing, and indeedincreasing, transfection efficiencies. Furthermore, using the method ofthe present invention, transfection efficiencies can be tightlyregulated by altering such parameters as frequency, pulse duration andpulse number.

[0013] The increase in DNA transfection efficiency is observed only ifthe muscle is electrically stimulated immediately, or shortly after theDNA injection. Thus, the semipermeable quality of the tissue induced bythe stimulation is reversible. Moreover, it is dependent on currentthrough the muscle; activity induced through the nerve does not affecttransfection efficiency.

[0014] Once transfected, the muscle cells are able to express theproteins coded by the nucleic acid. Therefore, the transfection methodof the present invention can be used, for example, to transfectexpression vectors for genetic immunization (i.e., DNA vaccines). In oneembodiment, rabbits were transfected with a plasmid containing the cDNAfor rat agrin. The transfected muscles produced and secreted agrinprotein. Nineteen days post-transfection, rabbit serum containedsignificant antibodies against rat agrin.

[0015] In a second embodiment, mice and rats were transfected using themethod of the present invention with one or more of three differenteukaryotic expression vectors containing the coding sequences forDH-CNTF, an agonistic variant of human ciliary neurotrophic factor,AADH-CNTF, an antagonistic variant of human ciliary neurotrophic factorand sec-DHCNTF, a secreted form of DH-CNTF. The muscles were either notelectrically stimulated or stimulated immediately after DNA injection.Blood was collected at various time points and the antibody titersdetermined. In both rats and mice, electrical stimulation immediatelyafter DNA injection led to approximately 5 to 10-fold higher antibodytiters than simple DNA injection.

[0016] The transfection method of the present invention can also be usedto systemically deliver proteins to treat diseases. In one preferredembodiment, a DNA plasmid harboring the erythropoietin (EPO) gene wasinjected into skeletal muscle and stimulated according to the method ofthe present invention. Controls were either not stimulated ortransfected with a control vector not harboring the EPO gene. After 14days, only the mice transfected with EPO according to the method of thepresent invention displayed an increased hematocrit indicating thetransfected muscles were able to produce and secrete into the bloodstream substantial amounts of EPO.

[0017] Non-nucleic acids may also be transfected by the method of thepresent invention. In one embodiment, rhodamin conjugated dextran wasinjected into the muscle followed by electrical stimulation. Three tofive days later the muscles were frozen in liquid nitrogen and sectionedon a cryostat. Fluorescence was observed in cells injected andstimulated, indicating the rhodamin conjugated dextran was able to enterand remain in the muscle cells.

[0018] In order to reduce pain that may be associated with the method ofthe present invention, a local anesthetic can be injected at the site oftreatment prior to or in conjunction with the injection of DNA. Forexample, in one embodiment of the current invention, DNA may be mixedwith Marcain, a local anesthetic, followed by electroporation.

[0019] These and other objects and advantages of the present inventionwill become apparent upon reference to the accompanying drawings andgraphs and upon reading the following detailed description and appendedclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The file of this patent contains at least one drawing executed incolor. Copies of this patent with color drawing(s) will be provided bythe Patent and Trademark Office upon request and payment of thenecessary fee.

[0021] A more particular description of the invention briefly describedabove will be rendered by reference to the appended drawings and graphs.These drawings and graphs only provide information concerning typicalembodiments of the invention and are not therefore to be consideredlimiting of its scope.

[0022]FIG. 1—graphically illustrates the method of deliveringpharmaceutical drugs and DNA into skeletal muscle of the presentinvention.

[0023]FIG. 2—is a graphical illustration of an electrical stimulationdelivered according to the method of the present invention.

[0024]FIG. 3—illustrates whole mounts of muscles which have beeninjected with 50 μl of RSV-Lac Z Plasmid DNA solution at a concentrationof 1 μg/μl. Muscles in 3 a and 3 b were taken out 15 days after DNAinjection. Muscles in 3 c and 3 d were taken out 7 days after DNAinjection. All muscles are pairs from the same rat.

[0025]FIG. 4—pictures a whole muscle and a 1 mm slice of a transfectedmuscle. Dark stain indicates o-nitrophenyl-b-D-galactopyranoside (ONPG)that has been catalyzed by β-galactosidase in the muscle to yield a darkprecipitate. Arrows illustrate muscle fibers that were successfullytransfected using the method of the present invention.

[0026]FIG. 5—includes mean number of transfected fibers from each groupof skeletal muscles shown in FIG. 3.

[0027]FIG. 6—is a bar graph illustrating mean transfected fibers ofmuscles from several different experiments and several different batchesof DNA grouped together. In columns marked SOL S and EDL S the muscles(16 in each group) have been stimulated directly after the injection ofDNA. In columns marked SOL NS and EDL NS the muscles (10 in each group)have been stimulated by the nerve, not stimulated at all or stimulateddirectly 10 minutes before the DNA injection.

[0028]FIG. 7—is a graph illustrating the number of skeletal musclefibers transfected versus the log of the stimulation frequency. Theduration of the stimulation train was kept constant at 1 second.

[0029]FIG. 8—is a photograph of transfected muscles from which data inFIG. 7 were generated.

[0030]FIG. 9—illustrates the results achieved when whole mounts ofmuscles were transfected according to the method of the presentinvention using two different electrodes.

[0031]FIG. 10—is a graph illustrating the number of skeletal musclefibers transfected with increasing frequency compared to increasingpulse number.

[0032]FIG. 11—is a graph illustration of the number of skeletal musclefibers transfected versus the number of pulses at constant frequency.

[0033]FIG. 12—is a graph illustrating mean luciferace activity versusthe number of pulses.

[0034]FIG. 13—is a graph illustrating the voltage dependency of thestimulation method of the present invention. FIG. 13a illustrates theluciferace activity of muscle stimulated with varying volts. FIG. 13billustrates the mean luciferace activity of muscles stimulated with anamplitude above 13 volts and below 5 volts.

[0035]FIG. 14—is a graph illustrating the effect of pulse duration onthe transfection efficiency.

[0036]FIG. 15—is a bar graph illustrating a comparison of transfectionefficiencies for varying pulse durations and pulse numbers.

[0037]FIG. 16—is a bar graph illustrating the effect of DNAconcentration on transfection efficiency.

[0038]FIG. 17—is a photograph of transfected muscles illustrating damagecaused by stimulation and regeneration of the muscle after a shortperiod of time. FIG. 17a illustrates an injected muscle that was notstimulated. FIG. 17b illustrates muscle damage following musclestimulation. FIG. 17c illustrates muscle stimulated under harsherstimulation conditions. FIG. 17d illustrates that muscles stimulatedunder the conditions of muscles in 17 c are completely regenerated andrepaired after 14 days. FIG. 17e illustrates muscles transfected withgreen fluorescent protein (GFP). FIG. 17f illustrates that transfectedfibers can bee seen in the vicinity of the damaged area.

[0039]FIG. 18—is a photograph of cells stained with anti-agrinpolyclonal antibodies derived from a rabbit genetically immunized withan expression vector coding for rat agrin using the stimulationtechnique of the present invention.

[0040]FIG. 19—are graphs illustrating improved genetic immunization ofmice and rats using the stimulation technique of the present inventionversus naked DNA injection.

[0041]FIG. 20—is a photograph of muscles transfected withrhodamine-conjugated dextran and green fluorescent protein. Top:rhodamin fluorescence from rhodamine conjugated dextran. Middle: Thesame section as above but with filters revealing GFP fluorescence.Bottom: hematoxilin and eosin staining of a neighboring section.

[0042]FIG. 21—are graphs illustrating relative amounts of specificsubtypes of antibody reactive with given antigens at four and eightweeks after immunization.

[0043]FIG. 22—is a bar graph illustration mean luciferace activity inlymph nodes of mice after transfection of muscle with Luc cDNA.

[0044]FIG. 23—is a bar graph illustration of mean luciferace activity inlymph nodes of rats after transfection of muscle with Luc cDNA.

[0045]FIG. 24—are graphs illustrating IgG1 antibody levels in mice at 4,8, and 9 weeks after genetic immunization.

[0046]FIG. 25—are graphs illustrating IgG2a antibody levels in mice at4, 8, and 9 weeks after genetic immunization.

[0047]FIG. 26—are graphs illustrating IgG2b antibody levels in mice at4, 8, and 9 weeks after genetic immunization.

[0048]FIG. 27—is a bar graph illustrating luciferace activity in musclecells 5 days after transfection with Luc plasmid DNA.

[0049]FIG. 28—is a bar graph illustrating mean luciferace activity inmouse muscles after a second immunization with 85B and luciferace cDNA.A low value indicates a strong cellular immune response and efficientkilling of transfected cells.

[0050]FIG. 29—are graphs illustrating antibody levels after proteinimmunization. A high level of IgG1 indicates a humoral immune response.

[0051]FIG. 30—are graphs illustrating antibody levels in mice eightweeks after genetic immunization with low electrical field strength.

[0052]FIG. 31—are graphs illustrating the responses of T-cells fromimmunized animals to the indicated peptides.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0053] The present invention is directed to a novel method forincreasing the permeability of skeletal muscle tissue and other cellsresiding therein, thus allowing pharmaceutical drugs, proteins, andnucleic acids to enter or transfect the cells. The method of the presentinvention passes a predetermined amount of electrical current throughthe skeletal muscle tissue. Unlike previously described electroporationmethods, however, the parameters of the method of the present inventionare unique, particularly with respect to the low field strength used andthe amount of damage that occurs. Other parameters such as the number oftrains, frequency, pulse number and pulse duration can be varied inorder to regulate the amount of pharmaceutical drug, protein, or nucleicacid delivered.

[0054] As illustrated in FIG. 1, generally, skeletal muscle is exposedand a predetermined amount of a molecule is injected into the muscle. Inone embodiment the DNA is dissolved in 0.9% sodium chloride (NaCl). Theexact solvent, however, is not critical to the invention. For example,it is well known in the art that other solvents such as sucrose arecapable of increasing DNA uptake in skeletal muscle. Other substancesmay also be co-transfected with the molecule of interest for a varietyof beneficial reasons. For example, P188 (Lee, et al. PNAS., 4524-8, 10,89 (1992)), which is known to seal electropermeabilized membranes, maybeneficially affect transfection efficiencies by increasing the survivalrate of transfected fibers.

[0055] With continued reference to FIG. 1, electrodes are placed on themuscle, about 1-4 mm apart, near the area where the molecule wasinjected. The exact position or design of the electrodes is not criticalso long as current is permitted to pass through the muscle fibersperpendicular to their direction in the area of the injected molecule.

[0056] Once the electrodes are in position, the muscle is electroporatedor stimulated. As illustrated in FIG. 2, the stimulation is delivered asa square bipolar pulse having a predetermined amplitude and duration. Inorder to optimize the transfection efficiencies, these parameters havebeen widely varied and transfection efficiencies compared. For example,the voltages have ranged from approximately 0 to 50 volts; the pulsedurations have ranged from 5 μs to 5 ms; the number of pulses haveranged from a single pulse to 30,000 pulses; and the pulse frequencywithin trains have ranged from 0.5 Hz to 1000 Hz.

[0057] The conclusion from these results is that so long as the fieldstrength is above about 50 V/cm, the other parameters may be varieddepending on the experimental conditions desired. While no upper limitwas detected, effective transfection efficiencies were observed withmuch higher field strengths. The field strength of the stimulation canbe calculated using the formula:

E=V/(2r ln(D/r)),

[0058] which gives the electric field between wires if D>>r. In theformula, V=voltage=10 V, D=distance between wire centers 0.1-0.4 cm,r=diameter of electrode=0.06 cm. See Hofmann, G. A. Cells in electricfields. In E. Neumann, A. E. Sowers, & C. A. Jordan (Eds.),Electroporation and electrofusion in cell biology (pp. 389-407). PlenumPublishing Corporation (1989). At 10 volts, the field strength isbetween 163 V/cm−43 V/cm (from 0.1 to 0.4 cm between electrodes,respectively). Because D is not much greater than r, it may be moreappropriate to use the formula for electric fields between largeparallel plates:

E=V/D

[0059] This gives a similar field strength of between 100 V/cm−25 V/cm(from 0.1-0.4 cm between electrodes, respectively). It will beappreciated that the field strength, as well as other parameters, areaffected by the tissue being transfected, and thus optimal conditionsmay vary. Using the parameters given in the present invention, however,optimal parameters can be easily obtained by one skilled in the art.

[0060] As illustrated in FIGS. 3 and 5-8, the method of the presentinvention dramatically increases the efficiency of drug and DNA deliveryinto skeletal muscle. In one embodiment, rat soleus or EDL muscles wereinjected with DNA plasmid containing the β-galactosidase gene (lac Z).The β-galactosidase gene yields a protein capable of converting acolorless substrate into a blue substrate that can be visually analyzedor measured spectrophotometrically. FIG. 3 depicts representative soleusand EDL muscles that have been transfected with β-galactosidase geneusing various stimulation parameters.

[0061]FIG. 3a illustrates the improved DNA delivery efficiency of soleusand EDL muscles that have been transfected according to the method ofthe present invention. Soleus and EDL muscles (n=3) were firstdenervated by transecting the sciatic nerve. This was done to eliminateany influence of nerve-induced activity that arguably could contributeto the increased transfection efficiency observed. Three dayspost-denervation, the muscles were injected with the P-galactosidasegene as described above. After the DNA injection, the muscles wereeither untreated or, immediately after the DNA injection, the muscleswere stimulated according to the method of the present invention.

[0062] Fifteen days after DNA injection the soleus and EDL muscles wereanalyzed. As illustrated in FIG. 3a, muscle cells that were stimulatedimmediately after DNA injection (bottom panels) contain more blueproduct indicating that more β-galactosidase gene was introduced intothe muscle cells. The transfection efficiency was quantitated bycounting the muscle fibers in a 1 mm cross section of the muscle thatcontained blue product as illustrated in FIG. 4. As illustrated by thebar graph in FIG. 5a, soleus muscle transfected using the method of thepresent invention showed a 47-fold increase over muscles that were notstimulated. Similarly, EDL muscle transfected using the method of thepresent invention showed a 12-fold increase over muscles that were notstimulated.

[0063] To determine whether nerve activity affected the transfectionefficiency, the method of the present invention was performed oninnervated (sciatic nerve not transected) and denervated (sciatic nervetransected) soleus and EDL muscles as described above. As illustrated inFIG. 3b, fifteen days after DNA injection both innervated and denervatedmuscles produced a generous quantity of blue product indicating highefficiency transfer of the β-galactosidase gene. As illustrated in FIG.5b, quantitation of transfected muscle fibers confirms high efficiencytransfection of both innervated and denervated muscles.

[0064] To rule out the possibility that the increased transfectionefficiency observed was due to muscle activity, direct stimulation ofthe sciatic nerve was compared to stimulation of the muscle (n=5). Ifthe increased transfection efficiency was due to muscle activity, thetransfection efficiency in muscles stimulated via the nerve should yieldsimilar efficiencies as direct muscle stimulation. As illustrated inFIG. 3c, direct nerve stimulation did not significantly increasetransfection efficiencies compared to direct muscle stimulation. Asillustrated in FIG. 5c, in both soleus and EDL muscles a 10-foldincrease in transfection efficiency was observed with direct musclestimulation.

[0065] As illustrated in FIG. 3d, the increased efficiency is transient,consistent with electroporation. Muscles stimulated directly after DNAinjection display significantly more blue dye than muscles that werestimulated prior to DNA injection. In fact, muscles that were stimulateddirectly after DNA injection displayed transfection efficiencies between10- and 25-fold greater than muscles that were stimulated 10 minutesprior to DNA injection (FIG. 5d).

[0066]FIG. 6 summarizes the results of the present invention. Musclesfrom several different experiments and several different batches of DNAare grouped together. In columns marked SOL S and EDL S the muscles (16in each group) have been stimulated directly after the injection of DNA.in columns marked SOL NS and EDL NS the muscles (10 in each group) havebeen stimulated by the nerve, not stimulated at all, or stimulateddirectly 10 minutes before the DNA injection.

[0067] The electrical stimulator used for the experiments wasmanufactured by FHC (Brunswick, Me. 04011). Both Pulsar 6 bp and thePulsar 6 bp-a/s stimulators have been used. The Pulsar 6 bp-a/s deliversa maximal voltage is 150 V and a maximal current of 50 mA. The maximalvoltage that can be delivered requires a resistance between theelectrodes of greater than 3000 ohms. The stimulators have been operatedat constant voltage mode. Because of the low resistance in the muscle,the voltages have been lower as discussed in the Examples below. In allexperiments the current has been maintained at 50 mA.

[0068] It will be appreciated by one skilled in the art that numerousother electrode configurations can be employed. For example, FIG. 9illustrates the results obtained using two different electrodesconfiguration. The electrode shown in (A) was placed perpendicular tothe muscle fibers. It consisted of a silver wire with diameter (d) of0.6 mm, (C) (this is the electrode which was used in all experimentsexcept in (B)). One electrode was placed on each side of the muscle. Ashort segment in the middle third of the muscle is positive for the LacZ staining (A), indicating localized expression. In (B) a 1.5 cmelectrode made from an insulated silver wire was used (d=0.3 mm).Insulation was removed from short segments (0.5-1.0 mm) along the wireat 2 mm intervals (D). The electrode was penetrated into the muscle inparallel with the muscle fibers. One of the two wires of the electrodewas penetrated into the muscle parallel with the muscle fibers. Thesecond wire was placed on the muscle surface, also parallel with thefibers. Both types of electrodes (FIGS. 9c and 9 d) gave a similarnumber of transfected fibers (approximately 250). Using the longerelectrode in parallel with the muscle fibers, however, gave a more widespread staining, indicating a transfection along a longer segment of thefibers and/or increased transfection.

[0069] Muscles were stained for Lac Z in whole mounts by methods wellknown in the art. After staining, the pictures were taken with thebluest side of the muscle up. Thereafter the muscle was cut in threepieces as seen in FIG. 2. The number of blue fibers in about 1 mm thickslice from the middle of the muscle were counted (fibers transfecteddistally or proximally from the slice are therefore not counted). Inorder to count the transfected fibers, the slices were dissected intosmaller bundles so single fibers could be distinguished under adissection microscope.

[0070] In four (4) muscles the pSV40-luc construct was used. It wasinjected into the soleus muscle, 3 days after the muscles were removedand luciferace activity was measured using the Promega Luciferace AssaySystem (Daviset et al., 1993). Uninjected EDL from the same rats wereused as control.

[0071] It will be appreciated that any nucleic acid can be used with themethod of the present invention, for example, plasmid DNA, linear DNA,antisense DNA and RNA. In one preferred embodiment, the nucleic acid isa DNA expression vector of the type well known in the art. Generally, anexpression vector contains a promoter operably linked to a DNA moleculethat codes for the protein of interest followed by a termination signalsuch as a polyadenylation signal. Other elements required for bacterialgrowth and proper mammalian processing may be included, such as theβ-lactamase coding region, an f1 origin and ColE1-derived plasmidreplication origin. Similar constructs containing a DNA coding region ofinterest can be constructed by one skilled in the art.

[0072] As illustrated in the examples below, molecules other thannucleic acids can be delivered to the muscle using the technique of thepresent invention. In one embodiment, rhodamin conjugated dextraninjected into the muscles and stimulated according to the method of thepresent invention was able to enter muscle cells. In addition, nucleicacid and proteins can be simultaneously introduced into anelectroporated muscle. In one embodiment, the large T-antigen nuclearlocalization signal was mixed with a plasmid containing the DNA codingregion for Lac Z. The large T-antigen nuclear localization signal is aprotein that binds DNA and facilitates its transport into the nucleus ofa cell. In other systems, large T-antigen nuclear localization signalhas been shown to increase transfection efficiency. Using the method ofthe present invention, large T-antigen nuclear localization signal alsoincreased the transfection efficiency of Lac Z indicating that theprotein was able to bind the DNA and enter the muscle cell.

[0073] The method of the present invention can be used to drive theimmune response of an animal in a specific direction. For example, DNAencoding for an antigen was administered to a group of mice according tothe method of the invention. After four and eight weeks, serum wascollected from the mice and antibodies analyzed by ELISA. The mice had ahigh level of IgG2a antibodies that reacted with the antigen, indicatingthat a strong cellular immune response was induced. When animals wereimmunized according to the method, increased number of CD8+ and CD4+T-cells that secreted interferon gamma was measured with ELISPOT afterstimulation with peptides specific for MHC I and II binding. Thisdemonstrate a strong cellular immune response as well as induction ofTh-1 cells. When animals were given a boost injection in combinationwith a reporter gene a reduced expression of reporter gene indicating astronger cellular immune response was observed in animals that had beenimmunized according to the present invention.

[0074] Another example is to drive the immune response in the otherdirection with preferential stimulation of the humoral branch of theimmune system. When protein was used in accordance with the presentinvention, higher IgG1 and IgG2b antibody titers could be detected. Whenthese protein-treated animals were given a boost injection of protein incombination with a reporter gene, increased expression of the reportergene was observed. These results indicate that immunization with proteinin combination with electrical stimulation had altered the immuneresponse in such a way as to make the animals tolerant to the antigen.Alternatively, such immunization may stimulate an immune reaction thatis not efficient in killing muscle cells. This method may be useful forthe treatment of various autoimmune diseases in which a strong cellularimmune response causes or contributes to the disease.

[0075] Without being bound by any particular theory, one mechanism toexplain these results could be that other cells in addition to skeletalmuscle cells can be transfected by the method of the present invention.For example, animals were intramuscularly injected with Luc cDNA. Themuscles were electrically stimulated shortly after the injection.Animals were sacrificed at two and seven days, and their spleens andlymph nodes removed and analyzed for luciferace activity. As shown inTable 5 below and FIGS. 22 & 23, a large increase in luciferace activityin the lymph nodes and spleen was found. These findings indicate thatimmune cells residing within the muscle are transfected by the method ofthe present invention.

[0076] Local anaesthetics are frequently used in medical procedures toreduce the pain and anxiety caused by the procedure. Marcain (2.5 mg/mlfrom Astra, bupivacain hydroclorid) is one such local anaesthetic.Marcain may be mixed with DNA to reduce the possible discomfort ofmuscle stimulation associated with electroporation. As seen in FIGS. 24through 27, the administration of Marcain had no significant effect oneither the resulting immune response or the transfection efficiency. Forthis procedure, a high concentration of Marcain was used. However, itwould be appreciated by those of skill in the art that otherconcentrations of Marcain and other anaesthetics can be used withoutdeparting from the nature of the present invention.

[0077] One may transfect the cells residing in the skeletal muscle usinglow electrical field strength, i.e., less than approximately 100 V/cm.In certain embodiments, cells within the muscle are transfected with afield strength of at least about 5 V/cm. In certain other embodiments,cells within the muscle are transfected with a field strength of atleast about 10 V/cm. For example DNA encoding an antigen wasadministered according to the method of the invention and the muscle wasstimulated with a field strength of about 10 V/cm to about 25 V/cm. Asseen in FIG. 30, a large increase in antibodies against the antigen weredetected for low voltage stimulation as compared to injected naked DNA.One might also use much greater field strengths. For example, one couldtransfect cells within muscle using field strengths in the range of fromapproximately 10 V/cm to approximately 300 V/cm. In certain embodimentsof the present invention, one could transfect cells within muscle usingfield strengths of from approximately 12 V/cm to approximately 175 V/cm;from approximately 125 V/cm to approximately 233 V/cm; or fromapproximately 10 V/cm to approximately 233 V/cm.

[0078] One skilled in the art would appreciate that a boost injection(immunization) given subsequent to the first immunization is likely toenhance the immune response further. This can be done withelectroporation or with other immunization strategies. For example,animals have been immunized with plasmid DNA and then later with acertain virus encoding the same antigen in order to obtain a furtherincrease in the cellular immune response. See, e.g., Schneider et al.,Nature Medicine 4:397 (1998). The boost injection may be given soonafter the initial immunization (i.e., within a few days or a week). Oneskilled in the art would also appreciate that booster immunizations maybe given many years after the first injection. In certain embodiments ofthe present invention, boost injections are given at a time from abouttwo weeks to about four months after the initial immunization. Incertain preferred embodiments, boost injections are given at a time fromabout one month to about two months after the initial immunization.

[0079] Without being bound by theory we have several possibilities forwhy EP enhances the immune response; first EP enhances expression of theantigen, second it enhances expression in other cells that are laterfound in lymphnodes or spleen, third it seem to enhance the musclescells ability to present antigen (MHC class I staining), fourth;electrical stimulation will cause muscle activity that is likely toincrease the lymphflow from the muscles (f.ex. low voltage stimulation).The mechanism are probably due to many of these factors in combinationand the contribution of each and one of them will depend on the type ofimmune response being induced.

EXAMPLES

[0080] The following examples are given to illustrate variousembodiments which have been made of the present invention. It is to beunderstood that the following examples are not comprehensive orexhaustive of the many types of embodiments which can be prepared inaccordance with the present invention.

Example 1 Stimulated Versus Unstimulated Muscles

[0081] Transfection efficiencies were determined by injecting skeletalmuscles with the pSV40-luc reporter construct into the soleus muscle.Three days after injection, the muscles were removed and luciferaceactivity was measured using the Promega Luciferace Assay System(Madison, Wis.) according to manufacturer's protocols. Unstimulated EDLmuscles from the same rates were used as control. The data are shownbelow in Table 1. TABLE 1 Stimulated Unstimulated Stimulated (RelativeUnstimulated luciferace- (Relative luciferace Percent Muscle activity)activity Increase Soleus animal I 34.40 1.950 1664% Soleus animal II21.50 0.250 8500% EDL animal I 0.045 EDL animal II 0.046

Example 2 Transfection Efficiency Versus Frequency

[0082] Rats were injected with 50 μ1 of 1 mg/μ1 of a plasmid carryinglac Z gene. Immediately following injection, electrodes were placedbetween 2-3 mm apart and the muscle was stimulated with the followingstimulation parameters: voltage=30 volts; pulse duration=0.2 ms (total0.4 ms, bipolar); trains=30, 1 second on 1 second off for 1 minute.Transfected fibers were counted from a 1 mm slice from middle of muscle.The number of transfected fibers is shown below in Table 2 andillustrated in FIG. 7. These data also illustrate that the method of thepresent invention transfects more than just surface muscle fibers;muscle fibers several cell layers deep are also transfected. TABLE 2TRANSFECTION EFFICIENCY VERSUS FREQUENCY Mean Frequency (TransfectedPercent (Hz) Fibers) Increase with Stimulation 0 22 — 1 83 277% 10 153595% 100 215 877% 1000 315 1332%

Example 3 Transfection Efficiency Versus Pulses

[0083] Soleus muscles of Wistar rats (200-270 grams) were injected with50 μg of RSV luciferace DNA plasmid in 50 μl 0.9% NaCl. Shortly afterinjection, the muscles were electrically stimulated using the followingparameters: 1000 Hz, between 0-1000 bipolar pulses of 200 μs duration ineach train were applied to the muscle 30 times over a period of 1minute.

[0084] Muscles were removed 3 days after transfection and frozen inliquid nitrogen. Cryostat sections were taken from the of the musclesand stained with Hematoxolin, Eosin and Safran (see Example 9). Theremaining pieces were homogenized as described in Example 4 below. Asillustrated in FIGS. 10-12, transfection efficiency increased with thenumber of pulses delivered to the muscle.

Example 4 Determining the Effect of Voltage on Transfection Efficiency

[0085] EDL and soleus muscles of Wistar rats (245-263 grams) wereinjected with 25 μg of RSV driven luciferace plasmid DNA in 50 μl 0.9%NaCl. Shortly after injection, the injected muscles were electricallystimulated using the following parameters: 100 Hz, 100 bipolar pulses ineach train of 200 μs duration, voltage varied from between 0 to 47.5.Muscles were removed 4 days post injection and stimulation, homogenizedin Promega (Madison, Wis.) luciferace assay buffer and luminescence wasmeasured according to manufacturer's protocols. Macintosh computer and aLabWiev acquisition program were used to capture the first voltagepulses. Recordings were done in parallel with the stimulationelectrodes. The voltage measurements were done manually on prints as theaverage of the maximal voltage of 10 pulses approximately 100 ms afteronset of stimulation.

[0086] As illustrated in FIG. 13a, there was a pronounced increase intransfection efficiency with increased voltage. As illustrated in FIG.13b, under the conditions of this experiment, muscles stimulated with 13volts or higher showed 40-fold greater luciferace activity compared tomuscles stimulated with 5 volts or less.

Example 5 Determining Optimal Pulse Duration

[0087] Soleus DNA plasmid containing the β-galactosidase gene in 50 μl0.9% NaCl. Shortly after injection, the muscles were electricallystimulated using the following parameters: 100 Hz, 25 volts, 100 bipolarpulses in each train having pulse durations ranging from 5-200 μs. Thenumber of transfected fibers were counted in a 1 mm thick section fromthe middle of the muscle under a dissection microscope. A second set ofrats were injected with 25 μg of RSV-driven luciferace plasmid DNA in 50μl 0.9% NaCl and electrically stimulated with the same parameters asabove except that the pulse durations were varied from 50-2000 μs. Asillustrated in Table 3 below and FIG. 14, under these stimulationparameters, the optimal pulse duration ranged from about 50 μs to about200 μs. This method can be used to optimize the pulse duration of otherstimulation parameters. TABLE 3 TRANSFECTION EFFICIENCY VERSUS PULSEDURATION Pulse Transfected Pulse Luciferace- Duration Fibers Durationactivity (μs) (Mean (μs) (Mean) 0 — 0 52.7 5 51 50 631 20 107 200 536 50228 500 348 200 272 2000 194

Example 6 Current versus number of pulses

[0088] Soleus muscles of six Wistar rats (178-193 grams) were injectedwith 50 μg of DNA plasmid containing the β-galactosidase gene in 50 μl0.9% NaCl. Shortly after injection, the muscles were electricallystimulated as described above except that the pulse duration was varied.The following electroporation parameters were compared: (1) 100 pulsesof 50 μs duration versus 1 pulse of 5000 μs; and (2) 10 trains of 100pulses of 50 μs versus 10 pulses of 5000 μs. Muscles were removed 14days later and sectioned on a cryostat. Cross sections were stained aspreviously described. The number of transfected fibers were counted. Asillustrated in FIG. 15, longer pulse durations result in highertransfection efficiency.

Example 7 DNA Concentration

[0089] EDL muscles of six Wistar rats (178-193 grams) were injected witheither 1 μg/μl or 5μ/μl of DNA plasmid containing the P-galactosidasegene in 50 μl 0.9% NaCl. Shortly after injection, the muscles wereelectrically stimulated with 30 trains of 100 pulses of 200 μs durationor not stimulated at all. Muscles were removed 14 days later andsectioned on a cryostat. Cross sections were stained as previouslydescribed and transfected fibers were counted. As illustrated in FIG.16, greater transfection efficiencies were obtained with higher DNAconcentrates.

Example 8 Large T Antigen Nuclear Localization Signal

[0090] Wistar rat muscles were injected with DNA plasmid containing theβ-galactosidase gene containing a 100:1 molar excess of large T-antigennuclear localization signal. This has been shown in other transfectionstudies to improve the transfection. See, Collas et al. Transgenic Res.6:451-8 (1996). The muscle were stimulated with 10 trains of 100 pulsesof 50 μs duration. The muscles containing the large T-antigen nuclearlocalization signal had the highest number of transfected fibers.Specifically, the muscle co-transfected with large T-antigen nuclearlocalization signal had 100 and 38 transfected fibers versus 7.3 and 4.7for the muscles transfected only with DNA, respectively. These dataillustrate that transfection efficiencies can be aided by mixing the DNAwith non-nucleic acid molecules. In addition, this data illustrates thatnon-nucleic acid molecules can also be delivered to the muscle using theelectroporation techniques of the present invention. No improvement wasseen in cells that were not stimulated following injection.

Example 9 Muscle Damage Resulting from Stimulation

[0091] Muscles from Example 3 that were sectioned and stained to assessthe muscle damage from electroporation. As illustrated in FIG. 17a, somedamage can occur with injection alone, although the majority ofunstimulated muscles were undamaged. In muscles stimulated with 300pulses, more damage was observed (FIG. 17b). As illustrated in FIG. 17c,muscle stimulated with 30 trains of 1000 pulses displayed greaterdamage, indicating that damage is proportional to the extent ofstimulation. FIG. 17d illustrates that muscles stimulated under theconditions of muscles in 17 c are completely regenerated and repairedafter 14 days.

[0092] In another muscle which got the highest amount of stimulation (30trains of 1000 pulses), plasmid DNA encoding green fluorescent protein(GFP), was also included. FIG. 17e illustrates muscles transfected withGFP. Transfected fibers can bee seen in the vicinity of the damaged area(FIG. 17f). Transfected regenerating fibers were never observed in crosssections 3 days after electroporation.

Example 10 Genetic Immunization of Rabbits

[0093] A female rabbit (4.5 kg) was injected into the right femuralisrectus with 2 milliliters of 1 μl/μl of DNA plasmid containing the ratneural agrin cDNA driven by the CMV promotor (Cohen et al. MCN, 9,237-53 (1997)). The first milliliter was injected equally in ten placessuperficial in the muscle followed by 10 trains of 1000 pulses deliveredat a frequency of 1000 Hz. The second milliliter was placed further downin the muscle. To test the rabbit serum, rat muscles and COS cells weretransfected with the same construct. Muscles were taken out 5 days aftertransfection and the COS cells were stained 4 days after transfection.

[0094] Bleeds were collected at days 0, 19, 50, 81 and 106 and diluted1:100 and 1:1000. After 19 days the bleed contained enough antibody inthe serum to give a weak staining of transfected fibers when diluted1:10. As a positive control the monoclonal antibody (mAb) AG-86 wasused. See Hoch et al. EMBO J, 12 (13): 2814-21(1994). Preimmune serumdid not show any staining of transfected fibers. Later bleeds all hadagrin antibodies in the serum. Bleed collected at day 50 or latercontained sufficient antibodies to stained sections at a dilution of1:1000.

[0095]FIG. 18a illustrates the agrin transfected COS cells stained withantiserum from immunized rabbit (diluted 1:100) and fluoresceinconjugated secondary antibody. COS cells were stained first fixing thecells in 1.5% paraformaldehyde for 10 minutes, followed by a 30 minutewash with phosphate buffered saline (PBS). The cells were then blockedwith 0.2% bovine serum albumin, triton X-100, 0.1% in PBS 0.1M, for 4minutes. Serum diluted in same solution was added to the cells andallowed to incubate for 20 minutes. Cells were wash for 4 minutes in PBSand incubated with the secondary antibody (Cappel, 55646) for 10 minutesfollowed by a PBS wash. Mouse primary mAb Agr-86 was included in thesame antibody mixture and rhodamin conjugated secondary antibody (SigmaT-5393, St. Louis, Mo.) was used at a dilution of 1:100. FIG. 18billustrates the same cells stained with mAb Ag-86/rhodamin conjugate.These data illustrate the potential of the technique of the presentinvention for genetic immunization or DNA vaccine technology.

Example 11 Genetic Immunization of Mice

[0096] Groups of two-month old male Sprague Dawley rats were inoculatedbilaterally in the EDL and soleus muscles with a total of 200 micrograms(4×50 microliters of a 1 mg/ml solution of DNA in saline) of threedifferent eukaryotic expression vectors containing the cytomegalovirusimmediate early promoter (CMV) and the coding sequences for thefollowing proteins: DH-CNTF, an agonistic variant of human ciliaryneurotrophic factor (Saggio et al. EMBO J. 14, 3045-3054, 1995);AADH-CNTF, an antagonistic variant of human ciliary neurotrophic factor(Di Marco et al. Proc. Natl. Acad. Sci. USA 93, 9247-9252, 1996);sec-DHCNTF, a secreted form of DH-CNTF. The muscles were either notelectrically stimulated or stimulated immediately after DNA injectionusing 30 trains of 100 or 1000 square bipolar pulses (duration 200microseconds; amplitude setting 150 V, effective voltage ˜25 V) each,delivered at a frequency of 1000 Hz with a two second interval betweensuccessive trains.

[0097] Groups of two-month old male CD1 mice were inoculated bilaterallyin the quadriceps muscles with 100 micrograms (2×50 microliters of a 1mg/ml solution of DNA in saline) of sec-DHCNTF plasmid, with or withoutelectrical stimulation of the muscle immediately after DNA injection.Stimulation conditions were 10 trains of 1000 square bipolar pulses(amplitude setting 150 V) delivered at a frequency of 1000 Hz with a twosecond interval between successive trains.

[0098] Blood was collected from the retroorbital sinus at selected timepoints and serum was prepared and stored at −20° C. The presence ofanti-CNTF antibodies in rat and mouse sera was determined by ELISA.Microtiter plates coated with recombinant human CNTF were incubated withserial dilutions of sera, followed by alkaline phosphatase-conjugatedantibody against rat or mouse IgG (Pierce). The plates were thenincubated in the presence of p-nitrophenyl-phosphate and the absorbanceat 405 nm was determined using a microplate reader. Antibody titers weredefined as the dilution of serum producing an absorbance reading equalto 50% of that obtained with a saturating concentration of anti-CNTFantiserum.

[0099] The results are shown in FIG. 19. Titers could not be averagedwith precision, due to the fact that some animals did not developdetectable amounts of antibody. Data are therefore presented forindividual animals, with a value of 1:100 representing a low orundetectable antibody titer (reciprocal titer ¾ 100). The results weresimilar for all plasmids used, as well as for rats and mice, as depictedin FIG. 19. Similar results were also obtained in both rats and micewith another plasmid encoding an unrelated viral protein (data notshown). In both rats and mice, electrical stimulation immediately afterDNA injection led to approximately 5 to 10-fold higher antibody titersthan simple DNA injection. This was true for stimulation with both highand low numbers of pulses. These results demonstrate that theelectroporation method increases the efficiency of DNA-mediatedimmunization.

Example 12 Secreted Proteins with Systemic Biological Activity

[0100] Fifty micrograms (50 microliter of a 1 mg/ml solution in 0.9%NaCl) of a eukaryotic expression plasmid (CMV-EPO) containing the cDNAof mouse erythropoietin under the control of the cytomegalovirusimmediate early promoter was injected in the left quadriceps muscle ofthree-month old 129xBalb/C female mice. In five mice (group 1), themuscles were electrically stimulated immediately after DNA injectionusing 10 trains of 1000 square bipolar pulses of 200 microsecondsduration, with an interval of 2 seconds between successive trains. Thefrequency of the trains was 1000 Hz and the amplitude set at 150 V(effective voltage ˜25 V). In another group of 5 mice (group 2) themuscles were not stimulated after DNA injection. As a control, a groupof 4 mice (group 3) was injected with a plasmid (CMV-GFP) containing thecoding sequence for green fluorescence protein under the control of theCMV promoter, followed by electrical stimulation at the same conditionsas group 1. Group 4 consisted of 5 mice injected only with salinesolution without electrical stimulation.

[0101] Blood was collected from the retroorbital sinus at selected timepoints and hematocrit was measured by centrifugation in capillary tubes.Serum samples were analyzed for the presence of EPO using a commercialELISA kit (R&D Systems). The results are shown in Table 4. In all groupsof mice, except those that were injected with the EPO construct andelectrically stimulated immediately thereafter, circulating EPO levelswere below the limit of detection of the ELISA kit (<15 mU/ml). Incontrast, mice injected with the EPO construct and electricallystimulated had significantly elevated serum EPO levels 5 days afterinjection (average of approximately 50 mU/ml). The serum concentrationof EPO remained elevated for up to 28 days following DNA injection(latest time point examined; data not shown). These levels of EPOproduced an increase in hematocrits, which rose from 46.2% prior toinjection to 70.0% and 76.7% at 14 and 28 days after DNA injection,respectively. These values were significantly different from thoseobtained with both control groups (groups 3 and 4) and from those ofmice injected with the EPO expression vector without electricalstimulation of the muscle (group 2). Indeed, the latter had hematocritlevels not significantly different from those of the control groups (seeTable 4). These results demonstrate that the electroporation method issuperior to simple DNA injection both in terms of the expression levelsof a secreted protein and in producing a biological effect mediated bythe secreted protein. TABLE 4 EPO SERUM CONCENTRATIONS AND ACTIVITY Day2 Day 5 Day 14 Mouse mEPO mEPO mEPO No. HCT % (mU/ml) HCT % (mU/ml) HCT% (mU/ml) Group 1 CMV-EPO 7 45 ND ND 55.7 71 72.4 Stimulated 8 48 ND ND54.6 68 5.3 9 47 ND ND 59 75.5 48.7 10 44 ND ND 62.2 69.5 62.9 11 47 NDND 7.9 66 22.4 Avg. 46.2 47.9 70.0^(abc) 48.3 Stand. Dev. 1.6 Group 2CMV-EPO 12 45 ND ND ND 50 <15 No stimulation 13 45 ND ND ND 50 <15 14 NDND ND ND 48 <15 15 46 ND ND ND 49.5 <15 16 44 ND ND ND 52 <15 Avg. 45 NDND ND 49.9 <15 Stand. Dev. 0.8 Group 3 CMV-GFP 2 ND ND ND ND 43.5 <15Stimulated 3 ND ND ND ND 48 <15 5 ND ND ND ND 48 <15 6 ND ND ND ND 48<15 Avg. ND ND ND ND 45.9 <15 Stand. <15 Dev. 1.8 Group 4 CMV-EPO 17 45ND ND <15 45.5 ND 18 45 ND ND <15 49 ND 19 43 ND ND <15 48 ND 20 45 NDND <15 51.5 ND 21 50 ND ND <15 47 ND Avg. 45.6 ND ND <15 48.2 ND Stand.Dev. 2.6 2.3

Example 13 Delivery of Non-nucleic Acid Molecules

[0102] Muscles were injected with 50 μl of a mixture of GPF plasmid DNA1 μg/μ1 and 2 μg/μl rhodamin-conjugated dextran (10 kD from MolecularProbes). Three to 5 days later the muscles (n=6) were frozen in liquidnitrogen and sectioned on a cryostat. As illustrated in FIG. 20,stimulated muscles (bottom) were transfected with rhodamin-conjugateddextran (top) and GFP (middle). As further illustrated, the same musclefibers were transfected with both GFP and rhodamin-conjugated dextran.These data indicate that non-nucleic acid molecules can be delivered tomuscle cells using the technique of the present invention.

Example 14 Materials and Methods for Genetic Immunization

[0103] The materials and methods listed below were employed throughoutthe Examples that follow, i.e., Examples 15-24, except as otherwiseindicated.

[0104] Protein Purification. Both of the mycobacteria secreted proteinsMPB70 and 85B were isolated and purified from culture fluid ofMycobacterium bovis BCG Tokyo and BCG Chopenhagen respectively, growingon Sauton media. PBS pH 7.4 was used to resuspend freeze-dried aliquotsof the purified proteins to appropriate concentration for immunizationor ELISA plate coating.

[0105] Plasmids. The following antigen-expressing plasmids were used inthese experiments: CMV70 which is the M bovis MPB70 protein encodingsequence inserted into the pcDNA3, a mammalian expression vector fromInvitrogen (Carlsbad, Calif.) with a CMV promoter (from Hewinson et.al., Central Veterinary Laboratory, Surry, UK).

[0106] 85b, which contains the mycobacterium tuberculosis gene encoding85B without the mycobacterial signal sequence inserted into thev1Jns-tPA vector from Merck. In this plasmid, the bacterial gene ispreceded by the promoter intron A of the first immediate early antigenIEI of CMV (85b from K. Huygen, Pasteur Institute of Brussels, Belgium).

[0107] Likewise, 85a is expresses the mycobacterium tuberculosis geneencoding the 85A protein, and a different form of 85b that it isinserted into the VR1020 vector from VICAL (from K. Huygen, PasteurInstitute of Brussels, Belgium).

[0108] Plasmids from transfected E. Coli cultures were amplified andpurified by using Genomed Jetstar purification kit, and by Aldevron DNAPurification Service to GMP standards. The purity of our DNA constructswere confirmed by enzyme digestion and agarose-EtBr electrophoresis. Theconcentration was measured by the 280/260 nm absorption ratio. The stocksolution was stored at −20° C. until needed.

[0109] The luciferase reporter gene used in these experiments containeda CMV promoter (VR-1255 from VICAL).

[0110] Luciferase assay. The assay was performed using the kit developedby Promega, with the organs in question homogenized and added to theassay buffer and purified by centrifugation. Activity was with aTD-20/20, Luminometer from Turner Designs (Sunnyvale, Calif., USA).

[0111] Animal experiments. Six- to eight-week-old female B6D2 mice orBalbC mice were anesthetized and used in accordance with Nonvegian rulesfor animal experiments.

[0112] Protein immunization. Mice were immunized subcutaneously with 100μl equal amount of proteins (MPB70 or 85B) at a concentration of 1 mg/mlin PBS sonicated with Incomplete Freunds Adjuvant (Behringwerke AG,Marburg, Germany).

[0113] BCG vaccination/immunization. Mycobacterium bovis BCG (Moreau)was harvested from cultures grown in Satoun medium and washed twice withPBS buffer. The spun-down bacteria were homogenized carefully with PBSto an approximate concentration of 200 mg/ml. 100 μl of this suspensionwas injected subcutaneously into the mice.

[0114] DNA injections and immunization with electrical stimulation (EP).Intramuscular injections were given with a 28-gauge insulin needle todeliver 0.5-50 μg of plasmid DNA in 50 μl of physiological saline toquadriceps in mice bilaterally (final concentration of DNA was 0.01,0.1, or 1 μg/μ1, for a total of 1, 10 or 100 μg DNA per mouse).Following the DNA injection, electrodes were placed on the skin todeliver an electric field at the site of DNA delivery. Theelectroporation was given as 8 trains of 1000 pulses delivered at afrequency of 1000 Hz. Each pulse lasted for 200 μs positive and 200 μsnegative for a total pulse duration of total 400 μs. The electricalfield strength varied with the change in resistance in the tissue ofeach animal, but the field strength was in the range of approximately25-35 V over approximately 2.5-3 mm, or from about 83 V/cm to about 140V/cm. Each train was delivered at two second intervals, with each trainlasting one second.

[0115] Serum sampling. Venous blood was taken from the mice after fourand eight weeks. Samples were left over night at 4° C. spun down andstored aliquoted at −20° C.

[0116] ELISA. ELISA were performed in Costar high-bind microtiter platescoated with native protein (85B or MPB70) 100 p.1 per well, 5 μ/μl inPBS with sodium azide (stable for months). Plates were stored at leastover night at 4° C. before use. Before use and between every step, theplates were washed 3 times with PBS+0.1% Tween 20. All incubations wereperformed at 37° C. for one hour, except the last developing step, whichwas performed at room temperature for 10 minutes. The assay steps wereas follows:

[0117] First, blocking was with PBS (without azide) containing 0.5% BSA,followed by application of serum samples diluted 27 or 64 times in PBSdilution buffer (0.2% BSA and 0.2% Tween 20. Biotinylatedsubtype-specific antibodies (anti-mouse IgG 1 (clone A85-1), anti-mouseIgG2a (R19-15), both Lou Rat IgG1, and IgG2b (R12-3) rat IgG2a) (allthree from Pharmingen) were added in a concentration of 0.5μ/ml dilutedin PBS dilution buffer. Streptavidin-HRP from Amersham diluted 1:1000 inPBS dilution buffer was then added. The amount of subtype-specificantibodies in serum were measured by OD at 405 nm after adding ABTSsubstrate in 0.1 M acetate buffer pH 4.0 with 3% H₂O₂.

[0118] Normalization of OD values in subtype ELISA. A positive controlstandard included in each ELISA microtiterplate was set to 1.0 (dividedby itself). All other values obtained were divided by the positivecontrol value, to be able to compare OD values from different microtiterplates within the same and different experiments.

EXAMPLE 15 Genetic Immunization with DNA Encoding Mycobacterial Antigens

[0119] B6D2 mice were selected for the experiment and divided into threegroups. One group received DNA plasmid and electrical stimulation (EP).The second group received only DNA. The third group consisted of controlanimals, which received only saline and electrical stimulation.

[0120] Each of the groups that received DNA were divided into subgroupsaccording of the dose and type of DNA injected. The total DNA dose usedin the mice was either 100, 10 or 1.0 μg in 100 μl saline (50 μl in eachmuscle). In FIG. 21, the symbols refer to different doses of DNA, witheach symbol representing the mean titer from a group of mice (5-7animals). Large symbols represent antibody titer from animals receiving100 μg DNA; medium-size symbols, 10 μg DNA; and small symbols, 1 μg DNA.Circles represents EP-treated animals and diamonds no EP. Filled squaresare from animals immunized with protein in IFA and plain lines (nosymbols) are from animals immunized with BCG bacila. Serum samples weretested by an ELISA assay designed for subtyping of antigen (MPB70 or85B) specific immunoglobulins. The average antibody titer is shown foreach group of animals as a function of optical density at 405 nm withserial dilution of serum samples collected at 4 and 8 weeks. The overallpattern is that EP-treated animals react with a significantly highertiter of immunoglobulins to the antigen encoded by the injected plasmidfor the three subclasses of immunoglobulin tested.

[0121] In mouse, a Th1 cellular immune response is indicated by elevatedserum concentration of antigen-specific immunoglobulins of subclass 2a.A humoral Th2 immune response is characterized by increasingantigen-specific IgGI and IgG2b antibodies in serum. Serum samples fromDNA-EP immunized mice contained elevated levels of IgG1, IgG2a and IgG2b(FIG. 21). This indicates that the animals react with both humoral andcellular immune responses. Compared with mice that have been immunizedwith M. bovis BCG or protein-IFA, our mice seem to have lower titers ofIgGi and IgG2b, but at the same or higher level with regard to IgG2a.These data indicate a strong Th1-associated cellular immune responsewhen the animals are injected with DNA and EP-treated.

[0122] The M. tuberculosis-specific secreted membrane protein MPB70tends to elicit a weaker immune response than the widely cross-reactingcommon mycobacterial antigen 85B. For 85b DNA, all three doses ofplasmid with EP give a high immunoglobulin response for the threeantibody subclasses tested. However, for mpb70 DNA, only the two highestdoses of plasmid injected give a high immunoglobulin response. When theanimals were injected with 1 μg of mpb70 DNA, we detected no immuneresponse. Animals that receive more than 1 μg 85b have almost the sameantibody titer as an animal that were given 100 μg. For mpb70, theresponse required a greater dose of DNA.

[0123] These results show that the dose of DNA injected into the animalscan be reduced at least 100-fold and still give the same or higherimmunoglobulin response against the antigen encoded. Without EP, none ofthe mpb70 animals react against the antigen, but for 85b, the twohighest doses of DNA give an immune response, although it issignificantly lower than the response in EP-treated animals. This mightbe because 85B is a common antigen, and the mice might have previouslybeen exposed to it.

[0124] We have also studied the gene transfection efficiency by stainingfor the encoded antigen in frozen cross sections of the quadricepsmuscle five days after plasmid DNA injection. By counting positivefibers in a defined area of the muscle section from EP-treated andnon-treated animals, we found a nearly hundred-fold increase in antigenexpression after EP (data not shown).

[0125] Muscle cells normally do not express either MHC class I or II ata detectable level. We have stained for both MHC I and II (mouse antiMHC class I from Pharmingen clone 34-2-12S, mouse anti MHC class IIclone 25-29-17, both were directly conjugated with FITC) in frozensections from the DNA-injected animals. With regard to MHC class I, wefind that neither muscle cells nor other cells in the area express MHC Iafter injection of saline followed by EP. With injection only of plasmid(no EP), the cells in muscle fasciae start to express MHC I at adetectable level. When the animals are injected with plasmid DNAfollowed by EP treatment, we see enhanced expression of MHC I in boththe fasciae and on muscle fibers in the area where there areplasmid-transfected and gene-expressing fibers. Characteristic stainingwas found, with positive MHC I circular staining in the periphery oftransfected fibers and their neighboring fibers, which is seen only inthe plasmid transfected area.

[0126] MHC class II was detected in the fasciae and in-between musclefibers only after plasmid injection followed by EP. HAS-staining showsthat mononucleated cells are recruited to the area after DNA injectionand EP treatment. The area in which we find MHC class II positive cellsseems to be co-localized to that in which we find mononucleated cellsafter HAS staining. Without being bound by any particular theory, thisco-localization may result from a combination of two factors. First, EPmay cause local damage to the muscle. Second, the expression of aforeign antigen encoded by the injected plasmid function as a strongsignal for recruitment of immune cells.

EXAMPLE 16 Transfection of Immune Cells Residing in Skeletal Muscle ofRats

[0127] Three rats were intramuscularly injected in the soleus muscle,surgically exposed, with 25 μg Luc cDNA dissolved in 50 μl of 150 mMsodium phosphate buffer pH 7.2. Following the injection, electrodes wereinserted into the muscle near the site-of the injection in two of therats, and electroporation given. The third rat received no electricalstimulation.

[0128] After two days, the spleens of the three rats were removed andanalyzed for luciferace activity. As shown in Table 5, the luciferaceactivity in the spleens of the rats that received electrical stimulationwas more than ten times greater than the activity the spleen of a ratthat did not receive electrical stimulation. These results indicate thattransfection of immune cells residing in muscle is increased byelectroporation. TABLE 5 Spleen Luc activity Fold increase after EP EP182 >10 EP2 83 >10 No EP 7.5

Example 17 Transfection of Immune Cells Residing in Skeletal Muscle ofMice

[0129] Eleven mice were intramuscularly injected into the quadricepswith 50 μg of Luc plasmid DNA dissolved in 50 μl of 150 mM sodiumphosphate buffer pH 7.2. In six of the eleven mice, the injection wasfollowed by electroporation.

[0130] After two days, the lymph nodes of the mice were removed andanalyzed for luciferace activity. As shown in FIG. 22, the luciferaceactivity in the lymph nodes of the mice that received electroporationexhibited significantly greater luciferace activity that in the micethat did not receive electroporation. These results indicate thatelectrical stimulation increases the transfection of immune cellsresiding in the muscle.

Example 18 Transfection of Immune Cells Residing in Skeletal Muscle ofRats

[0131] Six rats were intramuscularly injected in both surgically exposedsoleus muscles and EDL with 50 μg of Luc plasmid DNA dissolved in 50 μlof 150 mM sodium phosphate buffer pH 7.2. After the injection,electrodes were inserted near the injection site and electroporationgiven to both EDL and Soleus on the right side of the animal.

[0132] After seven days, the lymph nodes of the rats were removed andanalyzed for luciferace activity. Referring to FIG. 23, the luciferaceactivity in the lymph nodes draining the right-side muscles thatreceived electroporation was substantially higher than the activity ofthe lymph nodes draining the muscles on the left side that did notreceive electroporation. These results indicate that electricalstimulation increases the transfection of immune cells residing in themuscle at the time of electroporation. The cells travel to the lymphoidtissue, where they could play a role in inducing an immune response.

Example 19 Use of a Local Anesthetic During Genetic Immunization

[0133] Thirty-four mice were separated into groups of five to sevenmice. Each mouse was intramuscularly injected in the quadriceps asfollows. Group 1, saline+EP; group 2, mpb70 no EP; group 3 mpb70 and EP;group 4 mpb70+marcain but no EP; group 5 mpb70 marcain and EP. Finalconcentration in the solution containing DNA was 1 μg/μl dissolved in0.9% NaCl and group 4 & 5 also received 2.5 mg/ml Marcain in the DNAsolution. Both muscles in each animal were injected with 50 μ1 one ofthese solutions.

[0134] The electrical stimulation was delivered shortly after injectionto the muscles of selected mice and given near the site of injection.The sera were collected from the mice at four and eight weeks. A boostinjection (50 μg of mpb70) was given after 8 week. A final ELISA wasdone on serum collected after 9 weeks. Referring to FIGS. 24 though 26,ELISA analysis of the sera revealed no significant differences betweenthe genetically stimulated immune response in the animals that receivedMarcain and those that did not. Similar results are obtained with the85B construct.

Example 20 Use of a Local Anesthetic During MuscleTransfection/Electroporation

[0135] Mice were divided into two groups: group I consisted of fivemice, group 2 had six mice. Each mouse was injected with 50 μ1 with 25μg CMV Luc plasmid DNA dissolved in 0.9% NaCl in each left quadrisepsmuscle. The right muscle received the same amount of DNA but mixed withMarcain to a final concentration of 0.5 μg/μl DNA and 2.5 mg/mi Marcain.All muscles in mice in group 1 were not electroporated. All muscles ingroup 2 received electroporation.

[0136] After five days, the animals were sacrificed and the quadricepsremoved. The muscle were analyzed for luciferace activity. FIG. 27 showsthat animals that were injected with either CMV Luc plasmid DNA and orCMV Luc plasmid DNA and Marcain exhibited transfection at a high rateafter the electroporation treatment. These data demonstrate thatelectroporation performed with or without an anaesthetic results in thesame level of transfection.

Example 21 Protein Immunization with Electroporation

[0137] Six groups of mice were selected for use in the followingprotocol. Initial immunizations took place on day 0. Each mouse in thefirst group received an intramuscular injection of a saline solutionfollowed by electroporation. A second group of three mice received aninjection of 25 μg 85B protein and electroporation. Another group ofthree mice were injected with 25 μg 85B protein without electroporation.A group of five mice received 100 μg of 85b DNA in solution withoutelectroporation. Another group of five mice received 10 μg of 85b DNAand electroporation. Finally, a third group of five mice received 100 μgof 85b DNA and electroporation.

[0138] Eight weeks after the initial immunization, all animals weregiven a second immunization in which each animal in all six groupsreceived an intramuscular injection of a mixture of 25 μg of 85b DNA and1 μg Luc DNA, followed by electroporation. Five days later, the muscleswere removed and assayed for luciferace activity. In animals in which astrong cellular immune response was induced by the first immunization,one might expect to see a reduced luciferace activity compared to thoseanimals without a good induction of the cellular immune response.Without being bound by any particular theory, one might expect to seethis in two situations: when the cellular immune response is lacking orrepressed, or when the humoral branch of the immune system is activated,such that the second immunization with DNA primes the existing humoralresponse rather than a non-stimulated cellular response.

[0139] The results of this assay are shown in FIG. 28. The treatmenteach mouse received on day 0 is written on top of each bar. For example,“NaCl-85B” on top of the bar means the group received saline at day 0and the mixture of 85b and luciferase at week 8.

[0140] DNA without electrical stimulation did not have much effectcompared to saline. Both doses of DNA with electrical stimulation had aneffect, shown by the low luciferace activity. Without being bound by anyparticular theory, it appears that transfection at day 0 withelectroporation caused a cellular immune response that was rapidlymobilized and killed 85B/Luc-expressing fibers five days after the boostinjection at week 8.

[0141] However, with the protein, something else occurred as shown bythe large increase in luciferace activity. These results could be causedby a type of immune deviation. That is, the immune reaction has changedto a humoral type that was enhanced by the boost injection. Thishumoral-type immune response did not result in killing of thetransfected muscle fibers.

Example 22 Protein Immunization Followed by DNA Booster

[0142] Eight mice received NaCl and EP (control group), nine micereceived protein 85B (group, 85B+85b), five mice got protein MPB70(group, MPB70+mpb70) at day 0. The protein was given as an intramuscular injection of 20 vg purified protein 85B or MPB70 and electricalstimulated (right muscle only).

[0143] Eight weeks after the initial immunization, the animals weregiven a booster injection with DNA (35 μg in 50 μl 0.9% NaCl) encodingfor the corresponding protein antigen given in the first injection. Thecontrol group was split in two: four mice received mpb70 (groupNaCl+mpb70) and the other four received 85b group (NaCl+85b). Thesubsequent antibody response was measured five weeks later with ELISA.If the humoral response was stimulated/primed by the protein injection,one would expect to see a stronger increase in IgG1 antibodies afterimmunization with DNA.

[0144] Referring to FIG. 29, an elevated level of IgG1 was detected inthe mice that received the initial protein, either 85B or MPB70vaccination indicating that a humoral response was induced in these micecompared with mice that only received DNA. To demonstrate specificity ofthe assay, the ELISA was also done on serum from animals immunized witha different construct, hence the 85B ELISA was done on serum fromanimals that were previously immunized with the mpb70 plasmid (serve asa negative control).

Example 23 Low Voltage DNA Immunization

[0145] We have tested relatively low (less than 100 V/cm) electric fieldstrength, which could be used to avoid stimulation of and damage tosurrounding tissue. We used a low voltage, which we did not expect wouldhave much effect on transfection of muscle fibers. The low voltage,however, still had a significant effect on immunization.

[0146] Four groups of mice were selected for the following protocol.Each mouse was intramuscularly injected in the quadriceps as follows.The first group of six mice were injected with 0.9% saline and exposedto electroporation. Another group of six mice were injected with 100 μgmpb70 plasmid DNA dissolved in 0.9% NaCl and received noelectroporation. A third group consisting of seven mice were injectedwith 100 μg mpb70 plasmid DNA dissolved in 0.9% NaCl and receivedelectroporation at standard field strengths. A final group of seven micewere injected with 100 μg mpb70 plasmid DNA dissolved in 0.9% NaCl andreceived electroporation at lower field strengths.

[0147] The electrical stimulation was delivered shortly after injectionand given near the site of injection. Each mouse was electricallystimulated with 8 trains of 1000 pulses at 1000 Hz. Each pulse lastedfor 200 μs positive and 200 μs negative for a total pulse duration oftotal 400 μs. The electrical field strength varied with the change inresistance in the tissue of each animal, but the field strength for thestandard voltage level was in the range of approximately 50-70 V overapproximately 3-4 mm, or from about 125 to about 233 V/cm. The electricfield strength was from about 10 V/cm to about 25 V/cm at the lowvoltage electroporation. This low voltage stimulation caused strongmuscle contraction. Each train was delivered at two second intervalswith each train lasting one second.

[0148] After four and eight weeks, the sera of the mice was collectedand ELISA performed on sera. FIG. 30 shows the results of the eight weekELISA. The results were similar at four weeks, but are not shown. Thisexperiment was also done with a different antigen 85B with similarresults.

[0149] The eight week results show that low voltage stimulation enhancesimmune response compared to naked DNA injection. This enhanced immuneresponse could be due to the induced muscle activity or by transfectingcells other than muscle cells such as immune cells residing within themuscle.

Example 24 Increased Numbers of CD8- and CD4-Positive Cells AfterImmunization Using EP:

[0150] Twelve Balb/C mice were separated into four groups. Three micereceived 85a and EP, three received 85a without EP, three mice receiveda plasmid encoding P-galactosidase (β-gal, see previous Examples fordetails about construct) with EP, and three mice received β-gal withoutEP. Fourteen days later the spleens were removed from the animals. Cellswere isolated and treated according to standard ELISPOT procedures. SeeSchneider et al, Nature Medicine 4:397-402 (1998). Briefly, 1, 0.5 and0.25 million spleenocytes from each animal were placed in duplicates ofantibody-coated wells (anti-mouse INF-gamma mAb R4-6A2, hybridoma fromEuropean Collection of Animal Cell Cultures). Peptides (concentration 1μg/ml) were added to each test well. Control wells received irrelevantpeptide. After incubation overnight, plates were washed and incubatedfor 3 hours with a solution of 1 μg/ml biotinylated anti-mouse INF-gammamAb XMGI.2 (Pharmingen, CA), washed, and incubated for 2 hr with 50 μ1of a 1 mg/ml solution of Streptavidin-Alkaline-Phosphatase polymer(Sigma) at RT. Spots were developed by adding 50 μl of an alkalinephosphatese conjugated substrate solution (Biorad, Hercules, Calif.) andreactions were stopped by washing with water. Spots were countedelectronically.

[0151] The peptides used to stimulate spleenocytes from 85a immunizedanimals were: P-11, an epitope from 85A that specifically binds to MHCclass I and thereby stimulates CD8 positive cells (FIG. 31A); P-15, anepitope from 85A that specifically binds to MHC class H and therebystimulates CD4 positive cells (FIG. 31B). See Denis et al., Infect.Immun. 66:1527-1533 (1998) for details about the peptides.

[0152] The peptide used to stimulate spleenocytes from β-Gal immunizedanimals were AA-876-884 from E. Coli beta-galactosidase, this peptidespecifically binds to MHC class I and thereby stimulates CD8-positivecells. See FIG. 31C.

[0153] Results shown in FIG. 31 demonstrate an increased number of bothCD4 and CD8 positive T-cells when immunization is done in combinationwith EP. Hence, the cellular branch of the immune system is stimulated.

[0154] A high number of CD8- and CD4-positive T-cells is oftenassociated with good protection against many serious infectious diseasesin vaccinated humans. It is also believed to be important in protectionand the treatment of cancer.

[0155] The invention may be embodied in other specific forms withoutdeparting from its essential characteristics. The described embodimentsare to be considered in all respects only as illustrative and notrestrictive. The scope of the invention is, therefore, indicated by theappended claims rather than by the foregoing description. All changesthat come within the meaning and range of equivalency of the claims areto be embraced within their scope.

What is claimed is:
 1. A method of delivering a molecule to the skeletalmuscle of a mammal in vivo, comprising: (a) injecting a molecule intoskeletal muscle of a mammal, whereby a penetration site and a treatmentregion are created; (b) positioning electrodes spaced from saidpenetration site such that current traveling between the electrodespasses through the treatment region; and (c) electrically stimulatingthe muscle with an electrical current.
 2. The method of claim 1, whereinsaid current generates a field strength in the range of from about 25V/cm to less than 250 V/cm.
 3. The method of claim 1, wherein saidelectrical stimulation is delivered in the form of a single pulse. 4.The method of claim 3, wherein said pulse has a duration of betweenabout 50 μs and 5000 μs.
 5. The method of claim 1, wherein saidelectrical stimulation is delivered in the form of between about 2 to30,000 pulses.
 6. The method claim 5, wherein said pulses have a totalduration of between about 10 ms to 12,000 ms.
 7. The method of claim 6,wherein said pulses are delivered in the form of at least two trains. 8.The method of claim 7, wherein the frequency of said electricalstimulation is between about 0.5 Hz and 1000 Hz.
 9. The method of claim1, wherein said molecule is a nucleic acid.
 10. The method of claim 10,wherein said nucleic acid encodes a protein and said encoded protein isexpressed by muscle cells following step c.
 11. A method of delivering amolecule to the skeletal muscle of a mammal in vivo, comprising: (a)injecting a molecule into skeletal muscle of a mammal, whereby apenetration site and a treatment region are created; (b) positioningelectrodes spaced from said penetration site such that current travelingbetween the electrodes passes through the treatment region; and (c)electrically stimulating the muscle with a fixed electrical current thatresults in field strength that varies with tissue resistance, said fieldstrength varying from about 25 V/cm to less than about 250 V/cm.
 12. Themethod of claim 11, wherein said electrical stimulation is delivered inthe form of a single pulse.
 13. The method of claim 12, wherein saidpulse has a duration of between about 50 μs and 5000 μs.
 14. The methodof claim 11, wherein said electrical stimulation is delivered in theform of between about 2 to 30,000 pulses.
 15. The method of claim 14,wherein said pulses have a total duration of between about 10 ms to12,000 ms.
 16. The method of claim 15, wherein said pulses are deliveredin the form of at least two trains.
 17. The method of claim 16, whereinthe frequency of said electrical stimulation is between about 0.5 Hz and1000 Hz.
 18. The method of claim 11, wherein said molecule is a nucleicacid.
 19. The method of claim 18, wherein said nucleic acid encodes aprotein and said encoded protein is expressed by muscle cells followingstep c.
 20. A method of expressing a polypeptide in a mammal,comprising: (a) injecting one or more expression vectors into skeletalmuscle of a mammal, whereby a penetration site and a treatment regionare created, wherein (i) said vector contains a nucleic acid segmentthat encodes a polypeptide and (ii) said segment is under geneticcontrol suitable to express said polypeptide in cells of said mammal;(b) positioning electrodes spaced from said penetration site such thatcurrent traveling between the electrodes passes through the treatmentregion; and; (c) electrically stimulating the muscle with an electricalcurrent.
 21. The method of claim 20, wherein said current generates afield strength in the range of from about 25 V/cm to less than 250 V/cm.22. The method of claim 20, wherein said electrical stimulation isdelivered in the form of a single pulse.
 23. The method of claim 22,wherein said pulse has a duration of between about 50 μs and 5000 μs.24. The method of claim 20, wherein said electrical stimulation isdelivered in the form of between about 2 to 30,000 pulses.
 25. Themethod of claim 24, wherein said pulses have a total duration of betweenabout 10 ms to 12,000 ms.
 26. The method of claim 25, wherein saidpulses are delivered in the form of at least two trains.
 27. The methodof claim 26, wherein the frequency of said electrical stimulation isbetween about 0.5 Hz and 1000 Hz.
 28. A method of expressing apolypeptide in a mammal, comprising: (a) injecting one or moreexpression vectors into skeletal muscle of a mammal, whereby apenetration site and a treatment region are created, wherein (i) saidvector contains a nucleic acid segment that encodes a polypeptide and(ii) said segment is under genetic control suitable to express saidpolypeptide in cells of said mammal; (b) positioning electrodes spacedfrom said penetration site such that current traveling between theelectrodes passes through the treatment region; and (c) electricallystimulating the muscle with an a fixed electrical current that resultsin field strength that varies with tissue resistance, said fieldstrength varying from about 25 V/cm to less than about 250 V/cm.
 29. Themethod of claim 28, wherein said electrical stimulation is delivered inthe form of a single pulse.
 30. The method of claim 29, wherein saidpulse has a duration of between about 50 μs and 5000 μs.
 31. The methodof claim 28 wherein said electrical stimulation is delivered in the formof between about 2 to 30,000 pulses.
 32. The method of claim 31 whereinsaid pulses have a total duration of between about 10 ms to 12,000 ms.33. The method of claim 32, wherein said pulses are delivered in theform of at least two trains.
 34. The method of claim 33, wherein thefrequency of said electrical stimulation is between about 0.5 Hz and1000 Hz.